Processes and vectors for plastid transformation of higher plants

ABSTRACT

A process for producing multicellular plants, plant organs or plant tissues transformed on their plastome by the following steps is provided: (a) altering or disrupting the function of a gene in a plastid genome for producing a selectable or recognizable phenotype; (b) separating or selecting plants or cells having plastids expressing said phenotype; (c) transforming said plastid genome of said separated or selected plant, plant organ or plant tissue with at least one transformation vector having a restoring sequence capable of restoring said function; and (d) separating or selecting said transformed plant, plant organ or plant tissue having plastids expressing said restored function.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 National Phase Applicationof International Application Ser. No. PCT/EP02/00481, filed Jan. 18,2002 and published in English as PCT Publication No. WO 02/057466 onJul. 25, 2002, which claims priority to German Patent Application Ser.No. 101 02 389.8, filed Jan. 19, 2001, the disclosures of each of whichare incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention generally pertains to plant molecular biology andmore particularly pertains to novel methods for plastid transformation.

BACKGROUND OF THE INVENTION

According to generally accepted knowledge, two classes of cellorganelles, i.e. plastids and mitochondria, are derived from initiallyindependent prokaryotes that were taken up into a predecessor of presentday eukaryotic cells by separate endosymbiotic events (Gray, 1991). As aconsequence, these organelles contain their own DNA, DNA transcripts inthe form of messenger RNA, ribosomes, and at least some of the necessarytRNAs that are required for decoding of genetic information(Marechal-Drouard et al., 1991).

While, shortly after endosymbiotic uptake, these organelles weregenetically autonomous, since they contained all the elements necessaryto drive prokaryotic life, this autonomy was reduced during evolution bytransfer of genetic information to the cell's nucleus. Nevertheless,their genetic information is of sufficient complexity to make recentcell organelles an attractive target for gene technology. This isparticularly the case with plastids, because these organelles stillencode about 50% of the proteins required for their main function insidethe plant cell, photosynthesis. Plastids also encode their ribosomalRNAs, the majority of their tRNAs and ribosomal proteins. In total, thenumber of genes in the plastome is in the range of 120 (Palmer, 1991).The vast majority of proteins that are found in plastids are, however,imported from the nuclear/cytosolic genetic compartment.

Plastids can be Genetically Transformed

With the development of general molecular cloning technologies, itbecame soon possible to genetically modify higher plants bytransformation. The main emphasis in plant transformation was and stillis on nuclear transformation, since the majority of genes, ca. 26,000 inthe case of Arabidopsis thaliana, the complete sequence of which wasrecently published (The Arabidopsis Genome Initiative, 2000), is foundin the cell's nucleus. Nuclear transformation was easier to achieve,since biological vectors such as Agrobacterium tumefaciens wereavailable, which could be modified to efficiently enable nucleartransformation (Galvin, 1998). In addition, the nucleus is more directlyaccessible to foreign nucleic acids, while the organelles are surroundedby two envelope membranes that are, generally speaking, not permeable tomacromolecules such as DNA.

A capability of transforming plastids is highly desirable since it couldmake use of the enormous gene dosage in these organelles—more than 10000copies of the plastome may be present per cell—that bears the potentialof extremely high expression levels of trangenes. In addition, plastidtransformation is attractive because plastid-encoded traits are notpollen transmissible; hence, potential risks of inadvertent transgeneescape to wild relatives of transgenic plants are largely reduced. Otherpotential advantages of plastid transformation include the feasibilityof simultaneous expression of multiple genes as a polycistronic unit andthe elimination of positional effects and gene silencing that may resultfollowing nuclear transformation.

Methods that allow stable transformation of plastids could indeed bedeveloped for higher plants. To date, two different Methods areavailable, i.e. particle bombardment of tissues, in particular leaftissues (Svab et al., 1990), and treatment of protoplasts withpolyethylene glycol (PEG) in the presence of suitable transformationvectors (Koop et al., 1996). Both methods mediate the transfer ofplasmid DNA across the two envelope membranes into the organelle'sstroma.

One significant disadvantage of all multicellular plant transformationprocedures used today is the occurrence of marker genes in thetransgenic plants. These marker genes that are needed for the selectionof transgenic plant cells from a vast background of untransformed cellscode for antibiotic or herbicide resistance genes. Examples for plastidresistance genes are aadA conferring resistance to spectinomycin andstreptomycin (Svab & Maliga, 1993), or nptII conferring resistance tokanamycin (Carrer et al., 1993). As these marker genes are stablyintegrated into the genome together with the genes of interest (GOI),they will stay in the homoplastomic transgenic plants although they arenot required for GOI function. These remaining marker genes are a mainissue of criticism of plant biotechnology as they could theoreticallyincrease antibiotic resistance of pathogens or herbicide resistance ofweeds. Construction of a selection system which does not result in aresistance gene in the transgenic plant is, therefore, highly desirable(Iamtham and Day, 2000).

Another problem in plastid transformation is the shortage of selectablemarker genes available. The aadA gene is the only selectable marker genethat is used routinely (Heifetz, 2000), and the nptII gene is the onlyalternative that has been shown to function in higher plant plastidtransformation (Carrer et al., 1993). Since neither the aadA nor thenptII gene can be used universally, the number of higher plant speciesthat have been transformed in the plastome is still very low (Heifetz,2000). Plastid transformation in higher plants cannot at present beexploited to its full potential.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a simple, yet highlyversatile process for producing genetically stable multicellular plants,plant organs or plant tissues transformed in their plastome, which arefree of a foreign gene required for selection such as an antibiotic orherbicide resistance gene.

This object is achieved by a process for producing multicellular plants,plant organs or plant tissues transformed in their plastome by thefollowing steps:

-   -   (a) altering or disrupting the function of a gene in a plastid        genome for producing a selectable or recognizable phenotype;    -   (b) separating or selecting plants or cells having plastids        expressing said phenotype;    -   (c) transforming said plastid genome of said separated or        selected plants, seeds, cells or plastids with at least one        transformation vector having a restoring sequence capable of        restoring said function; and    -   (d) separating or selecting said transformed plants or cells        having plastids expressing said restored function.

Preferred embodiments are defined in the subclaims.

It is surprising that this new method is readily applicable tomulticellular plants, plant organs, or plant tissue since these containa plurality of plastids in each cell, which means that segregation ofgenotypes is required on the level of plastomes, the level of plastidsand the level of cells. It has been found that this new process ishighly efficient for the tissue of higher plants since segregationoccurs readily during growth. Separation is therefore simply possible byoptical inspection and manual manipulation in appropriate embodiments.In cases of inhibitor-supported selection (step (b)), the selectionprocess can be carried out rapidly since in the case of multicellularplants, plant organs or plant tissue the inhibitor does not need to beapplied throughout the whole regeneration process, but may be appliedonly initially. (Of course, as explained above, it is possible to avoidinhibitors completely.) This shows a close combination effect betweenmulticellularity and the method of transformation.

In the case of a transformation of a plant tissue by the process of theinvention, the consequences of alteration or disruption of a gene, whichmay frequently be lethal in the case of a single isolated cell if thisgene is of central importance e.g. for a metabolic pathway, aremitigated by the fact that a single transformed cell does not stand inisolation. Rather, it is part of a population of cells among whichmetabolites may be exchanged.

There are many different plastid genes which can be altered or disruptedfor the purposes of this invention. Such a gene should be important forplastid function in the sense of producing a selectable or recognisablephenotype upon alteration or disruption. Such a function may be anyfunction which is plastid encoded. Preferably, this function is directlyor indirectly involved in photosynthesis. Examples for functionsindirectly necessary for photosynthesis are any functions needed fortranscription and/or translation of plastid genes. Examples forfunctions directly involved in photosynthesis are any proteins which areessential, at least under selection conditions, for photosynthesis.

Preferably, said recognisable phenotype is easily discernable. Sincesaid function above is preferably directly or indirectly associated withphotosynthesis, an easily recognisable phenotype may be pigmentdeficiency, most preferably chlorophyll deficiency or alteredfluorescence. The transformed plant may then be grown heterotrophicallyand transformed plants, plant organs or plant tissue may be separated orselected for. Separation may be effected manually by optical recognitionof transformed tissue areas. Selection may be effected via inhibitorresistance based on a resistance gene introduced in step (a) of theprocess of this invention. Alternatively, inhibitor resistance may be aconsequence of said altered or disrupted function itself.

After reaching the homoplastomic state by segregation during severalrounds of regeneration, the transgenic plant, plant organ or planttissue is transformed a second time (step (c)), whereby the altered ordisrupted function is restored and the marker gene, if any, is removed.The transformed plants, plant organs or plant tissues having therestored phenotype, e.g. phototrophy, are subsequently separated orselected.

Additional sequences or genes of interest may be introduced in step (a)and/or step (c) e.g. for expressing a desired gene, for conferring auseful trait or for any other desired plastome modification.

Further, sequences introduced in step (a) and step (c) may togetherresult in an additional function. Examples for this embodiment includethe following: introducing sequences in step (a) and step (c) that codefor different subunits of a multi-subunit protein; providing regulatorysequences in step (a) or step (c) that make a coding sequence introducedin step (c) or step (a), respectively, expressible; introducingsequences in steps (a) and (c) that code for proteins of a biochemicalpathway; etc.

Specific examples of the function to be disrupted may be knock outs ofrpoA or rpoB. These genes code for the α and β subunit, respectively, ofthe plastid encoded plastid RNA polymerase. Plastids lacking these genesare not able to conduct photosynthesis, show an albino phenotype, andare not able to grow phototrophically. After restoration of rpoA or rpoBin the second round of transformation, the transgenic plants are able togrow phototrophically in light and show a green phenotype.

Another example of a target gene for step (a) may be a knock out ofycf3. This gene is not essential under normal light conditions (Ruf etal., 1997). Nevertheless, if a ycf3 knock out mutant is placed understrong light, it develops an albino phenotype and growth is repressedunder photosynthetic growth conditions. Plants with restored ycf3 geneare able to grow phototrophically under strong light conditions. So inthis case, the selection pressure for the second transformation can beadjusted by the light intensity. Therefore, in contrast to the exampleusing rpoA or rpoB, the second transformation can be carried out withgreen, normally growing plant mutants when kept under low lightconditions and selection pressure can be raised after a regenerationtime simply by increasing the light intensity. As the condition of theplant material is critical for transformation, this method is superiorto transformation of albino material.

Another example for the function to be altered or disrupted is a knockout of petA in the first round of transformation (step (a)). petAencodes a subunit of the cytochrome b/f complex. petA knock out mutantsshow a high chlorophyll fluorescence phenotype (hcf) and are not able toconduct photosynthesis. Therefore, they are not able to growphototrophically. The phototrophic growth in light is restored, whenpetA is restored in the second round of transformation (step (c)).

Definitions

The following definitions are given in order to clarify the meanings ofcertain terms used in the description of the present invention.

3′-UTR transcribed but not translated region of a (→) gene, downstreamof a (→) coding region; in (→) plastid (→) genes, the 3′-UTR a.o. servesto stabilise the mRNA against 3′ to 5′ exonucleolytic degradation 5′-UTRtranscribed but not translated region of a (→) gene, upstream of a (→)coding region; in (→) plastid (→) genes, the 5′-UTR contains sequenceinformation for translation initiation (ribosome binding site, (→) RBS)close to its 3′ end aadA (→) coding region of bacterial aminoglycosideadenyl transferase, a frequently used protein, that detoxifiesantibiotic (→) selection inhibitors spectinomycin and/or streptomycinchloroplast (→) plastid containing chlorophyll coding region nucleotidesequence containing the information for a) the amino acid sequence of apolypeptide or b) the nucleotides of a functional RNA; coding regionsare optionally interrupted by one or more (→) intron(s) desired gene,modified or newly introduced sequence: the purpose of a (→) (sequence)transformation attempt flank, DNA sequences at the 5′ and 3′ ends ofinserts in a (→) plastid (→) flanking region transformation (→) vector,which mediate integration into the target (→) plastome of sequencesbetween the flanks by double reciprocal (→) homologous recombination. Bythe same mechanism, sequences can be modified or removed from the target(→) plastome. Thus, the flanks of the (→) plastid (→) transformation (→)vector determine, where changes in the target (→) plastome are generatedby (→) transformation. gen xpr ssion process, turning sequenceinformation into function; in (→) genes encoding polypeptides, geneexpression requires the activity of a (→) promoter which initiates anddirects RNA polymerase activity, leading to the formation of a messengerRNA, which is subsequently translated into a polypeptide; in (→) genesencoding RNA, the (→) promoter- mediated activity of RNA polymerasegenerates the encoded RNA gene(s) nucleotide sequence(s) encoding allelements, which are required to secure function independently; genes areorganised in (→) operons, which contain at least one complete (→) codingregion in (→) genes encoding polypeptides, these elements are: (1) a (→)promoter, (2) a 5′ untranslated region ((→) 5′-UTR), (3) a complete (→)coding region, (4) a 3′ untranslated region ((→) 3′-UTR); in (→) genesencoding RNA, the (→) 5′-UTR and the (→) 3′-UTR are missing; in (→)operons consisting of more than one (→) coding region, two subsequentcomplete (→) coding regions are separated by a (→) spacer, and (→)promoter, (→) 5′-UTR, and (→) 3′-UTR elements are shared by the (→)coding regions of that (→) operon. genome Complete DNA sequence of acell's nucleus or a cell organelle hcf high chlorophyll fluorescence;hcf mutants show a characteristic photosynthesis deficient phenotypeheteroplastomic a (→) plastid or cell containing genetically differentplastomes plastid/cell homologous process leading to exchange, insertionor deletion of sequences due to recombination the presence of (→) flankswith sufficient sequence homology to a target site in a (→) genomehomoplastomic a (→) plastid or cell containing genetically differentplastomes plastid/c II ins rtion sit locus in the (→) plastome, intowhich novel sequences are introduced interg nic region sequences betweentwo (→) g nes in a (→) g nome; such region occur as (→) int r peronicregions or as (→) intraoperonic regions, in which case they are alsocalled (→) spacers intragenic region sequences inside a (→) gene intronsequence interrupting a (→) coding region organ a plant organ is astructure that serves a special biological function and consists of oneor more characteristic (→) tissues; examples are: root, stem, leaf,flower, stamen, ovary, fruit etc. operon organisational structure of (→)genes petA (→) coding region of the (→) plastid (→) gene for thecytochrome f protein involved in photosynthetic electron transportplant(s) organism(s) that contain(s) (→) plastids in its cells; thisinvention relates to multicellular (→) plants; these include the groupof gymnosperms (such as pine, spruce and fir etc.) and angiosperms (suchas monocotyledonous crops e.g. maize, wheat, barley, rice, rye,Triticale, sorghum, sugar cane, asparagus, garlic, palm tress etc., andnon-crop monocots, and dicotyledonous crops e.g. tobacco, potato,tomato, rape seed, sugar beet, squash, cucumber, melon, pepper, Citrusspecies, egg plant, grapes, sunflower, soybean, alfalfa, cotton etc.),and non- crop dicots as well as ferns, liverworths, mosses, andmulticellular green, red and brown algae. plastid(s) organelle(s) withtheir own genetic machinery in (→) plant cells, occurring in variousfunctionally and morphologically different forms, e.g. amyloplasts, (→)chloroplasts, chromoplasts, etioplasts, gerontoplasts leukoplasts,proplastids etc. plastome complete DNA sequence of the (→) plastidpromoter nucleotide sequence functional in initiating and regulatingtranscription RBS, DNA sequence element upstream of the (→) translationstart cod n of ribos mal binding a (→) coding r gion, that mediatesribosome binding and translation site initiation from the respective RNAtranscript; RBS elements are either part of (→) 5′-UTRs or of spacers.rpoA/B/C (→) coding regions of the (→) plastid (→) genes for the plastidencoded RNA-Polymerase (PEP) selection inhibitor chemical compound, thatreduces growth and/or development of non- transformed cells ororganelles stronger than that of transformed ones tissue a plant tissueconsists of a number of cells with similar or identical structure andfunction; cells in plant tissues are connected by plasmodesmata;examples are: callus, palisade parenchyma, spongy parenchyma, cambium,epidermis, pith, endosperm, phloem, xylem etc. transformation cloned DNAmolecule that was generated to mediate (→) vector transformation of a(→) genome; transformation process leading to the introduction, theexcision or the modification of DNA sequences by treatment of (→) plantsor plant cells including the use of at least one (→) transformationvector transgene DNA sequence derived from one (→) genome, introducedinto another one translation start sequence element, that encodes thefirst amino acid of a polypeptide codon translation stop sequenceelement that causes discontinuation of translation codon uidA (→) codingregion of bacterial β glucuronidase, a frequently used reporter proteinycf3 (→) coding region for a protein that is involved in PSI assembly;Δycf3 lines display a pale phenotyp and growth depression, whencultivated under standard light conditions (3.5–4 W/m²). Under low lightconditions (0.4–0.5 W/m²) the phenotype is much less severe.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Schematic view of vector pIC571.

FIG. 2: Schematic view of vector pIC554.

FIG. 3: Schematic view of vector pGEM-rpoA-del and targeted plastomeregion.

FIG. 4: Schematic view of vector pIC598 and targeted plastome region.

FIG. 5: Schematic view of vector pIC553.

FIG. 6: Schematic view of vector pKCZ-GFP.

FIG. 7: Schematic view of vector pIC526.

FIG. 8: Schematic view of vector pIC558 and targeted plastome region.

FIG. 9: Schematic view of vector pIC558.

FIG. 10: Schematic view of vectors pIC597, pIC599 and pIC600 andtargeted plastome region.

FIG. 11: Schematic view of vector pIC597.

FIG. 12: Map vector pIC577.

FIG. 13: Schematic view of vector pIC577 and targeted plastome region.

FIG. 14: Map of vector pIC637.

FIG. 15: Schematic view of vector pIC637 and targeted plastome region.

DETAILED DESCRIPTION OF THE INVENTION

Vectors of this Invention Provide a Visible Marker During Selection

Non-lethal inhibitor concentrations that do not kill plant cells butinhibit growth to a certain degree can be used for plastidtransformation. Only one or a few of the up to 10,000 plastome copiesper cell can be assumed to be recombinant after the initialtransformation event. Treating the cultured cells or tissues with alethal inhibitor concentration after transformation would not allow torecover heteroplastomic cells expressing a moderate resistance, which isdue to a low number of transformed plastome copies. Selection andsegregation leads to the occurrence of both wild-type and transplastomictissues. It is a major problem to discriminate between wild-type andtransgenic tissue during this process, because transformed plastids maymask wild type ones. Khan and Maliga (1999) used a fluorescentantibiotic resistance marker, comprising the aadA and GFP codingregions, to track segregation in plastid transformants under UV-light.In this invention, we present a visible marker for the transplastomictissue sectors which can be detected by the naked eye. The gradualprocess of sorting out wild-type and recombinant plastids can easily bemonitored and thus be accelerated. The appearance of green sectors onthe white background of the mutant phenotype can be easily detected.

Vectors of this Invention Provide Improved Regeneration Efficiency

Conventional chloroplast transformation strategies are based on theselection for resistance against an inhibitor, e.g. spectinomycin.Inhibitor application starts after transformation and is perpetuatedduring the whole process of repetitive regenerations which are necessaryto obtain a homoplastomic genotype. Inhibitor application has thedisadvantage to reduce the regeneration potential. Regeneration of wholeplants from single protoplasts or leaf pieces is a critical step inchloroplast transformation, particularly when extending the method tospecies, for which established and reliable regeneration protocols donot exist. It is a major advantage of this invention, that inhibitorsmay only have to be utilized during a short period after the firsttransformation. Using our novel methods, inhibitor application can beomitted during repetitive regeneration in order to achieve ahomoplastomic condition and during the complete second transformationstep.

Vectors of this Invention Allow Generation of Genetically StablePlastome Transformants

Homologous recombination in plastids is known to occur with highefficiency. As a consequence, undesired recombination events betweenregulatory elements of an antibiotic resistance marker and endogenousregulatory elements may lead to genetic instability (Eibl et al., 1999).After the second transformation step the transplastomic plant does notcontain any marker expression cassette. Consequently the finaltransformants contain fewer regions of homology than conventionalplastid transformants. The genetic stability of the transformants isincreased and undesired loss of sequences (Eibl et al., 1999) isavoided.

By this invention, a novel antibiotic-free, photosynthesis relatedselection system for chloroplast transformation of higher plants can beprovided. The new system utilizes visible markers and may be based onthe inactivation of genes like rpoA, rpoB, ycf3 or petA yielding a whiteor pale phenotype. In a second step, the respective deficient gene ofthe mutant line may be restored and one or more transgenes may beinserted. The resulting transplastomic plants may be free of anantibiotic resistance gene.

Other possible target functions for step (a) of the process of thisinvention are any plastid encoded functions which are directly orindirectly required for photosynthesis. Apart from the specificapplications described below, inactivation and restoration of numerousphotosynthesis related target genes, such as e.g. psbA may be usedaccording to this invention.

Plastid chromosomes encode four RNA polymerase genes, designated rpoA,B, C1 and C2, that resemble the three RNA polymerase core genes ofeubacteria. The genes for rpoB, C1 and C2 are arranged in an operon(transcribed by NEP, a nuclear encoded plastid RNA polymerase), whilethe gene for rpoA is located in a large cluster of genes that mainlyencode ribosomal proteins. Since the level of the sense transcript ofthe rpoA gene decreases in PEP (plastid encoded plastid RNA polymerase)deficient mutants (Krause et al., 2000), rpoA might be transcribed byPEP.

Deletion of rpoA, rpoB or rpoC1 from the plastid genome results in apigment-deficient phenotype (Allison et al., 1996; De Santis-Maciosseket al., 1999). The pigment-deficient ΔrpoA, ΔrpoB or ΔrpoC1 plants(white plants) are unable to grow photoautotrophically. However, ifmaintained on sucrose-containing medium to compensate for the lack ofphotosynthesis, they grow normally but at a reduced rate compared withwild-type plants.

ycf3 has recently been shown to be required for stable accumulation ofthe photosystem I (PSI) complex in tobacco (Ruf et al., 1997).Disruption of this gene leads to a conditional pigment-deficientphenotype in light. Homoplasmic ycf3 plants display a completely whitephenotype upon regeneration on drug- and phytohormone-free medium understandard light conditions (3.5–4 W/m²), while the phenotype is much lesssevere (light green) under low light conditions (0.4–0.5 W/m²).

A mutant plant phenotype which is called hcf (high chlorophyllfluorescence) is well known. This phenotype is due to a mutation inexpression and/or processing of photosynthesis related genes (eithernuclear or plastome encoded genes; Bock et al. 1994; Monde et al., 2000;Monde et al., 2000b). These mutants show a characteristic photosynthesisdeficient phenotype: impaired growth under greenhouse conditions, palegreen leaves, and high chlorophyll fluorescence (red fluorescence) underUV-light illumination. Hcf appears when the photosynthetic electrontransport chain is blocked (‘electron tailback’). One possibility toachieve a hcf phenotype is to inactivate the plastid petA gene, whichcodes for a subunit of the cytochrome b/f complex involved inphotosynthetic electron transport.

Taking advantage of the pigment or photosynthesis deficient phenotypesof, for example, Δrpo, Δycf3 or ΔpetA plants, a second round oftransformation may be performed using the first round pigment-deficienttransformants as a substrate to restore the deficiency, remove theselection marker of the first round, if any, and optionally introducesequences of interest simultaneously. Green plants can be recovered bydelivering a wild type gene into the plastome of pigment-deficientmutants. Therefore, such secondary transformants will regain the abilityof photosynthetic growth and display a green phenotype and/or normalchlorophyll fluorescence in case of the hcf phenotype. Thischaracteristic can be used to select transformed tissues. Thus noantibiotic selection is required in this second step. More importantly,selection markers used during the first round of transformation can beremoved in the second round, yielding marker-free transplastomic plants.

Plastid gene transformation is based on homologous recombination. Thiscan be achieved by using, in a transformation vector, flanking regionsof sufficient homology to target sites on the plastome, which iswell-known in the art. In the present invention, transformation may beperformed by any method known in the art. Presently, there are two suchknown methods, namely particle gun transformation and PEG-mediatedtransformation. In this invention, particle gun transformation ispreferred. Both steps (a) and (c) of the process of this invention mayalso be achieved by co-transformation, i.e. using more than onetransformation vector.

The teaching of this invention may be applied to any multicellularplant. Preferred plants are monocot and dicot crop plants. Specificexamples of crop plants are listed herein under item “plants” in section“Definitions”.

There are several possibilities for altering or disrupting the functionof a plastid gene in step (a) provided that a selectable or recognizablephenotype is produced. Examples include partial or full deletion of thecoding region of said gene or of a functional element required forexpression of said gene e.g. promoter, 5′-UTR, 3′-UTR, and start codon.The function of these elements may also be altered or disrupted byinsertion of a foreign sequence into these elements or the codingregion, by full or partial replacement of these elements by a foreignsequence or by insertion of a stop codon into the coding region. Theabove means may also be combined. If a resistance marker gene isintroduced in step (a), the marker gene is preferably used as saidforeign sequence. In step (a) any additional sequence of interest may beinserted concomitantly.

Step (a) of the process of this invention is preferably achieved bygenetic transformation. In an alternative embodiment, step (a) may occuror may have occurred by a spontaneous or induced mutation. This meansthat a plant (or plant organs or plant tissue) used for step (c) of thisinvention may be a natural mutant or a transgenic plant not obtainedaccording to this invention or for purposes of this invention, which ispredominantly the production of transgenic plants free of a marker gene.

In step (b) of the process of this invention, plants, plant organs orplant tissue having plastids expressing the phenotype of interest areseparated or selected for on medium supporting heterotrophic growth.Selection may be done using a selectable marker gene introduced in step(a) and a suitable antibiotic or inhibitor. Alternatively, the novelprocedures of this invention using photosystem I acceptor herbicides asdescribed in more detail below may be applied. In the latter case, noresistance gene has to be introduced in step (a). As described above,inhibitors or antibiotics do only have to be utilized during a shortperiod after the first transformation to support segregation and the useof such an agent can even be neglected totally. After growth and severalcell divisions, segregation leads to the formation of zones differing inpigment abundance or fluorescence. Tissue from such zones is separatedmanually and is used for further rounds of regeneration.

In step (c) of the process of this invention, the plastome of a plantobtained in previous steps is transformed with a vector having arestoring sequence capable of restoring the function altered ordisrupted in step (a). Said restoring may be achieved by several meansdependent on how alteration or disruption in step (a) was done. Insertedsequences may be removed, replaced sequences may be replaced again withthe original fully functional sequence, and deleted sequences may bereinserted. Concomitantly, a resistance gene inserted in step (a) may beremoved or its function may be destroyed and an additional geneticmodification of the plastome may be carried out or an additionalfunction may be introduced. Examples include introduction of anadditional sequence or gene of interest, introduction of several genes,elimination of a preexisting function or sequence etc.

In step (d), plants, plant organs or plant tissue having plastidsexpressing said restored function are separated or selected for onantibiotic-free medium. Selection may be achieved by at least partialphototrophic growth and transformed plants are recognizable by therestored phenotype. Upon growth, segregation leads to the formation ofzones of differing pigment abundance. Green zones are separated manuallyand used for further rounds of regeneration. In this invention, theconditions used in step (d) are preferably mixotrophic. This means thatthe carbohydrate content of the medium is lowered as much as possiblesuch that plastids containing transformed plastomes and cells containingtransformed plastids which have regained the ability for photosynthesishave a selective growth advantage under strong light. Such mixotrophicconditons may accelerate step (d).

Embodiment 1: Method for the Selection of Plastid Transformants Based onthe Inactivation and Restoration of the rpoA or rpoB Genes

For targeted disruption of rpoB gene function, the rpoB promoter and thestart codon may for example be replaced with the aadA marker gene oranother marker gene. Bombarded leaf tissue may be regenerated undertemporary selection on spectinomycin-containing medium in case of theaadA marker. Transformants display antibiotic resistance and initially agreen phenotype in light while still being heteroplastomic. Theseprimary transformants contain a mixture of both wild-type andtransformed chloroplast genomes. The green, heteroplastomic material istransferred to non-selective medium. Segregation leads to the occurrenceof white, mixed, and green sectors. Material from white sectors may besubjected to several additional rounds of regeneration on non-selectivemedium in order to obtain homoplastomic mutant transformants.

In the second transformation, the homoplastomic ΔrpoB plants may betransformed with a vector designed to reconstitute the rpoB gene, removethe marker gene and preferably introduce a gene (or genes) of interestat the same time. The treated leaf tissue may be regenerated underselection on sucrose-reduced medium (antibiotic free) under stronglight. Transformants which display a green phenotype and are able togrow photoautotrophically may be selected.

A disruption and reactivation of the rpoA gene may be achieved in asimilar way.

Embodiment 2: Method for the Selection of Plastid Transformants Based onthe Inactivation and Restoration of the ycf3 Gene

Disruption of ycf3 gene may be achieved by replacing the 5′-regulatoryelement and the first exon of ycf3 by a marker gene like the aadA markergene. The transformation vector may be introduced into tobacco plastidse.g. using the biolistic protocol or PEG-mediated transformation. Thebombarded leaf tissue, in case of the biolistic protocol, is regeneratedunder selection on medium containing an inhibitor or antibiotic,spectinomycin in case of the aadA gene. Transformants display inhibitorresistance and initially a green phenotype under standard lightconditions (3.5–4 W/m²) while still being heteroplastomic. These primarytransformants normally contain a mixture of wild-type and transformedchloroplast genomes. After transfer to antibiotic-free medium,segregation leads to the occurrence of yellow-white and green sectors(under standard light conditions; see above). Material from whitesectors may be subjected to several additional rounds of regeneration onnon-selective medium in order to obtain homoplastomic mutanttransformants. Besides having a pale, nearly white phenotype in light,the mutants show depressed growth. To obtain adequate material for thesecond transformation step, the mutant plant line may be transferred tolow light conditions. Under these conditions (0.4–0.5 W/m²), the plantsshow a much less severe phenotype and can yield suitable donor materiale.g. for particle gun transformation. In this second transformation, thehomoplastomic Δycf3 plants are transformed with a vector designed toreconstitute the ycf3 gene, remove the marker gene and preferablyintroduce a gene (or genes) of interest at the same time. The bombardedleaf tissue may be regenerated under selection on sucrose-reduced medium(antibiotic free) under strong light. Transformants, which display anormal green phenotype and are able to grow photoautotrophically, may beselected.

Embodiment 3: Method for the Selection of Plastid Transformants Based onthe Inactivation and Restoration of the petA Gene

For targeted disruption of the petA gene, the coding region may bereplaced with a marker gene, e.g. the aadA marker gene. Bombarded leaftissue, in the case of transformation by particle bombardment, may beregenerated under selection on antibiotic containing medium.Transformants display antibiotic resistance and initially a normal greenphenotype in light while still being heteroplastomic. These primarytransformants contain a mixture of wild-type and transformed chloroplastgenomes. After transfer to antibiotic-free medium, segregation may leadto the occurrence of sectors displaying the hcf phenotype, which can bedetected under UV illumination. Material from the mutant sectors may besubjected to several additional rounds of regeneration on non-selectivemedium in order to obtain homoplastomic mutant material.

In the second transformation, the homoplastomic ΔpetA plants may betransformed by bombardment with a transformation vector designed toreconstitute the ΔpetA gene, remove the marker gene and preferablyintroduce a gene (or genes) of interest at the same time. The bombardedleaf tissue may be regenerated under selection on sucrose-reduced medium(antibiotic free) under strong light. Transformants which display anormal green phenotype and are able to grow photoautotrophically may beselected.

Embodiment 4: Method for the Selection of Plastid Transformants Based onthe Inactivation and Restoration of the petA Gene, Whereby InactivationMutants are Selected by a Novel Procedure

This procedure may be used with all mutants that are photosynthesisdefective. Similar to embodiment 3, selection of plastid transformantsmay be based on the inactivation and restoration of the petA gene. Incontrast, the selection for ΔpetA mutants may be carried out on mediumcontaining a herbicide that requires active photosynthesis for efficacy,e.g. the herbicide Paraquat. Any complete inactivation of the petA genemay result in an increased resistance of the mutant plant line to such aherbicide compared to wild-type. Importantly, the introduction of anyantibiotic or herbicide resistance marker during the firsttransformation step can be omitted.

Vectors of this Invention Provide the Possibility to Introduce NovelFunctions During the First or the Second Transformation Step

Genes or sequences of interest may be introduced during the first or thesecond step of transformation or during both. Therefore, multiple genesor functional operons may be introduced into the target plant. Amongothers, this is of particular interest for the generation of newmetabolic pathways in transplastomic plants, as a significant number ofnovel genes and/or regulation factors may have to be integrated into theplastome.

Similarly, desired sequences may be introduced or removed e.g. in orderto manipulate plastid gene expression pattern.

Vectors of this Invention Provide the Possibility to Reuse SelectionMarkers

A further application of the two step strategy described in thisinvention is the possibility to reuse the same selection marker foranother transformation after removing it from the genome during thesecond step. Subsequently, it may be removed again and the process maybe repeated. This provides means for the insertion of a potentiallyunlimited number of genes or functional operons into the plastid genomeusing the same marker gene. This is an important step towards overcomingthe shortage of selection markers for plastid gene transformation.

Vectors of this Invention Allow the Introduction of Sequences ofInterest at Independent Loci

By using various combinations of the described methods (e.g. embodiment1, 2 and 3 using rpoA, ycf3 and petA, respectively, as target sites),the genes of interest can be introduced into different target sites ofthe plastome. The method described here will also function usinginactivation and restoration of other photosynthesis related targetgenes, such as e.g. psbA. Consequently numerous potential target sitesexist. Using homologous recombination, novel functions may also beintroduced at sites independent of marker genes.

Vectors of this Invention Provide Novel Selection Schemes

The aadA gene is the only selectable marker gene that is used routinely(Heifetz, 2000), and the nptII gene conferring resistance to kanamycinis the only alternative that has been shown to function in higher plantplastid transformation (Carrer et al., 1993). The vectors of thisinvention overcome the shortage of selectable marker genes. Novelselection inhibitors for plastid transformation that are described hereinclude paraquat, morphamquat, diquat, difenzoquat and cyperquat. Thesesubstances belong to the group of photosystem I acceptor herbicides(Hock & Elsner, 1995). They are not inhibitors of photosystem 1, butthey are reduced by photosystem I instead of ferredoxin and NADP.Autooxidation of reduced inhibitors then produces oxygen radicals whichare highly toxic. The toxicity of these herbicides therefore depends onlight and oxygen.

If the electron transport through photosystem I is interrupted either bydeletion of an essential gene of photosystem I or of the cytochrome b/fcomplex (for example petA) these mutant plants are more resistant tophotosystem I acceptor herbicides like paraquat than wild-type plants.Such herbicides may therefore be suitable selection agents for the knockout of these genes. Any albino will be insensitive to these inhibitors;thus any disruption that leads to photosynthesis deficiency could beselected for this way.

In one embodiment of the invention, step (d) may be assisted byinhibitor resistance. Inhibitor resistance may be achieved via stable ortransient introduction of an inhibitor or antibiotic resistance in step(c). Preferably, an inhibitor resistance introduced in step (c) istransient in order to allow the generation of selection marker freetransplastomic plants as an end result. An inhibitor resistance gene maybe removed by methods known in the art, e.g. as described by Fischer etal. (1996) or by Iamtham and Day (2000). Further, an inhibitor orantibiotic may be applied only initially in step (d) similarly asdescribed above for step (b). Omitting the inhibitor at a later stageallows loss of a resistance gene introduced in step (c). This embodimenttakes advantage of the regeneration of a discernible phenotype in steps(c) and (d) according to the principles of the invention, but theachievement of a homoplasmic state in step (d) may be made moreefficient.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Ausubel et al.,1999, Maniatis et al., 1989 and Silhavy et al., 1984.

Example 1 Construction of a Selection System Based on the Inactivationof the rpoB Gene

Construction of Transformation Vector pIC571 for the Inactivation of therpoB Gene

Leaves of tobacco plants were ground under liquid nitrogen and total DNA(Nicotiana tabacum L. var. petit havanna) was isolated using the Qiagen“DNeasy Plant Mini Kit”.

Using this total genomic DNA as a template the region of the tobaccochloroplast genome containing the rpoB and trnA7 genes was amplified byPCR. The following pair of oligonucleotide primers was used: p38 5′-AAGATG AAC CTG TTC CCA TG-3′ (SEQ ID NO:1) (annealing with plastomenucleotides 25967–25986; position numbers according to GenBank accessionnumber Z00044.1) and p39 5′-CAC TTC TTC CCC ACA CTA CG-3′ (SEQ ID NO:2)(annealing with plastome nucleotides 29616–29597). The PCR amplificationusing Taq-polymerase (Sigma) was performed as follows: 60 sec at 95° C.,1 cycle; 30 sec at 94° C., 60 sec at 55° C., 240 sec at 72° C., 32cycles; final extension at 72° C. for 10 min. The reaction products wereanalyzed by agarose gel electrophoresis. Only a single fragment could bedetected. It showed the expected size of 3.65 kbp. The fragment wasligated into vector pCR11 (Invitrogen) according to the protocol of thesupplier, yielding plasmid pCR rpoB01. The identity of the plasmidinsert was verified by sequencing (Toplab; Munich).

To inactivate the plastid rpoB-Operon, a selectable aadA marker cassetteshould replace the 5′-upstream region and the translation start of rpoB,represented by a 699 bp Ava I fragment (plastome postion 27508–28206).As a prerequisite, an additional Ava I restriction site in the multiplecloning site of plasmid pCR rpo01 had to be removed. This was done bycutting the plasmid with the enzyme Xho I, followed by a fill-inreaction using Klenow polymerase and nucleotides. The linear fragmentwas then religated and transformed into bacteria. As a consequence theresulting plasmid pCR rpoB ΔXho only contained the two Ava I sitesmentioned above.

Plasmid pCR rpoB ΔXho was digested with Ava I. The larger of the tworesulting fragments (6861 bp and 699 bp) was purified from an agarosegel using the Qiagen gel extraction kit. The resulting sticky ends ofthe 6861 bp fragment created by the Ava I treatment were converted intoblunt ends using Klenow enzyme and nucleotides. The resulting DNA wastreated with calf alkaline phosphatase (Roche Diagnostics GmbH,Mannheim, Germany) to suppress self ligation in the following step.

Finally, a 1412 bp Sma I fragment containing the aadA expressioncassette from vector pUC16SaadA-Sma (Koop et al., 1996) was ligated intothe 6861 bp Ava I fragment. The ligation products were transformed intobacteria. The plasmids of the resulting bacterial clones were analyzedfor the presence and the orientation of the aadA-insert. 7 positiveclones showed insertion of the aadA-cassette in sense direction comparedto the rpoB gene. The plasmid was designated pIC571 (pCR rpoB aadA-I)(FIG. 1). Large amounts of pCR rpoB aadA-I plasmid DNA were isolatedusing the Qiagen Plasmid Maxiprep kit.

Primary Transformation and Selection of Homoplastomic ΔrpoB Mutants

PEG mediated transmembrane DNA transfer into protoplasts is areproducible method for plastid transformation of higher plants (Goldset al., 1993; O'Neill et al., 1993). Protoplast regeneration wasrecently optimized according to Dovzhenko et al., 1998.

A. Protoplast isolation: Leaves from about 3 weeks old tobacco plants(Nicotiana tabacum cv. petit Havanna) were cut to 1 mm stripes andincubated overnight with 0.25% cellulase R10 and 0.25% macerozyme R10(Yakult, Honsha Japan) dissolved in F-PIN medium. Following standardfiltration, flotation and sedimentation procedures (Koop et al., 1996)protoplasts were resuspended in transformation medium, the total numberof protoplasts was determined, and the density was adjusted to 5×10⁶protoplasts per ml.

F-PIN medium (pH 5.8 (KOH), osmolarity: 550 mOsm): KNO₃(1012 μg/ml),CaCl₂.2H₂O (440 μg/ml), MgSO₄.7H₂O (370 μg/ml), KH₂PO₄ (170 μg/ml),NH₄-succinate (10 ml of 2M stock), EDTA-Fe(III) Na-salt (40 μg/ml), KJ(0.75 μg/ml), H₃BO₃ (3 μg/ml), MnSO₄.H₂O (10 μg/ml), ZnSO₄.7H₂O (2μg/ml), Na₂MoO₄.2H₂O (0.25 μg/ml), CuSO₄.5H₂O (0.025 μg/ml), CoCl₂.6H₂O(0.025 μg/ml), inositol (200 μg/ml), pyridoxin-HCl (2 μg/ml),thiamin-HCl (1 μg/ml), biotin (0.02 μg/ml), nicotinic acid (2 μg/ml),BAP (1 μg/ml), NAA (0.1 μg/ml), Polypuffer 74 (10 ml), sucrose (˜130 000μg/ml).Transformation medium (pH 5.8 (KOH), osmolarity: 550 mOsm): MgCl₂.6H₂O(3050 μg/ml), MES (1000 μg/ml), mannitol (˜80000 μg/ml).

B. Plastid transformation and protoplast embedding: 50 μg DNA(transformation vector pIC571), 7 μl F-PCN, and 100 μl (500,000 cells)of protoplast suspension were added to 125 μl 40% PEG solution, mixedcarefully and incubated for 7.5 min. Another 125 μl of F-PCN were added,mixed and incubated for 2 min. The volume was filled up to 3 ml (withF-PCN) and 3 ml of F-alginate medium was added. Alginate embedding inthin layers is performed by applying 625 μl of protoplast-alginatemixture to polypropylene grids laying on the surface of Ca²⁺-medium.After solidification grids were removed and placed upside down intoliquid F-PCN medium for equilibration (2×10 ml, 30 min each) and thentransferred to a new petri dish with 2 ml F-PCN. The embeddedprotoplasts were incubated in the darkness for the initial 20 hours,followed by a usual 16 h day/8 h dark cycle.

F-PCN medium (pH 5.8 (KOH), osmolarity: 550 mOsm): KNO₃(1012 μg/ml),CaCl₂.2H₂O (440 μg/ml), MgSO₄.7H₂O (370 μg/ml), KH₂PO₄(170 μg/ml),NH₄-succinate (10 ml of 2M stock), EDTA-Fe(III) Na-salt (40 μg/ml), KJ(0.75 μg/ml), H₃BO₃ (3 μg/ml), MnSO₄.H₂O (10 μg/ml), ZnSO₄.7H₂O (2μg/ml), Na₂MoO₄.2H₂O (0.25 μg/ml), CuSO₄.5H₂O (0.025 μg/ml), CoCl₂.6H₂O(0.025 μg/ml), inositol (200 μg/ml), pyridoxin-HCl (2 μg/ml),thiamin-HCl (1 μg/ml), biotin (0.02 μg/ml), nicotinic acid (2 μg/ml),BAP (1 μg/ml), NAA (0.1 μg/ml), Polypuffer 74 (10 ml), sucrose (˜20 000μg/ml), glucose (65 000 μg/ml).F-alginate medium (pH 5.8 (KOH), osmolarity: 550 mOsm): MES (1370μg/ml), MgSO₄.7H₂O (2500 μg/ml), MgCl₂.6H₂O (2040 μg/ml), mannitol(˜77000 μg/ml), alginate (24000 μg/ml).Ca²⁺-medium (pH 5.8 (KOH), osmolarity: 550 mOsm): MES (1950 μg/ml),CaCl₂.2H₂O (2940 μg/ml), mannitol (˜82000 μg/ml), agar, purified (10000μg/ml).

One week after transformation embedded protoplasts were transferred tosolid RMOP medium (see example 3) containing 500 μg/ml spectinomycin andstreptomycin each. Every 3 weeks the grids were transferred to freshmedium until no further regenerates appeared. First green regeneratesappeared after 5 weeks and were transferred to single petri dishes. Asexpected, primary ΔrpoB transformants displayed spectinomycin-resistanceand a green phenotype in the light while still being heteroplastomic. Inorder to amplify transformed plastid DNA molecules and to eliminatewild-type genomes, the transformant colonies were transferred to RMOPmedium without inhibitors. White sectors appeared after 3 to 5 weeks ofculture. Material from white sectors was further subcultured onnon-selective medium and subjected to 5 further cycles of regenerationin order to obtain homoplastomic mutant transformants. The resultinglines showed a white phenotype. The transplastomic lines were rooted andpropagated on solid VBW-medium (see examples) to obtain mutant plantmaterial for the secondary transformation.

Analysis by PCR and Southern Blotting

Leaves of the mutant ΔrpoB transplastomic plants were ground underliquid nitrogen and total DNA (Nicotiana tabacum L. var. petit havanna)was isolated using the Qiagen “DNeasy Plant Mini Kit”.

Plastid transformants were analyzed by PCR amplification. Total DNAisolated from regenerates of several independent lines were used astemplates for separate PCR reactions. Two sets of oligonucleotideprimers were used to analyze the transplastomic plants. oFCH59 5′-TGCTGG CCG TAC ATT TGT ACG-3′ (SEQ ID NO:3) (derived from the 5′ portion ofthe aadA coding region) and oFCH60 5′-CAC TAC ATT TCG CTC ATC GCC-3′(SEQ ID NO:4) (derived from the 3′ portion of the aadA coding region)were used to detect the presence of the aadA gene. p42 5′-ATT TGT AGTAGA AGG TAA TTG C-3′ (SEQ ID NO:5) (annealing with plastome nucleotides29081–29102) and oFCH60 were used to detect correct integration of theaadA gene.

Additional proof of correct integration and the homoplastomic genotypewas given by DNA gel blot analysis. Genomic DNAs isolated from sterilegrown plants were used for DNA gel blot analysis. The detailed procedurewas as follows: 3 μg of total plant DNA per analyzed plant were digestedwith the appropriate restriction enzyme and separated on a TAE agarosegel (0.8%). The DNA was denatured and transferred onto a positivelycharged nylon membrane (Hybond-N+, Amersham) as described in Ausubel etal. (1999). The filter was hybridized with digoxigenin-labeled probes inDIG Easy Hyb Buffer (Roche Diagnostics GmbH, Mannheim, Germany), andhybridization signals were detected using the DIG Luminescent DetectionKit (Roche). The membrane was exposed to an X-OMAT LS film at roomtemperature for 75 minutes. For preparation of a hybridization probe, a398 bp Sma I/Hind III fragment was excised from plasmid pCR rpoB01,purified from a agarose gel and labeled using the Dig probe labeling kit(Roche).

Construction of Transformation Vector pIC554 for Reconstitution of therpoB Gene

For a proof of principle of the selection system, a transformationvector was constructed, which reconstitutes the deletion of the rpoBregulatory region and introduces a marker restriction site at the sametime. The additional marker restriction site should allow todifferentiate between recombinant plastome fragments in the respectivearea and a potential selection of residual wildtype plastome copies (incase the mutant lines were not completely homplastomic).

Plasmid pIC571 (pCR rpoB01) was cut with Xma I. The ends of the linearfragment were converted into a blunt form using Klenow polymerase andnucleotides. The resulting DNA was religated and transformed intobacteria. Plasmids of the bacterial clones were screened for the absenceof the Sma I restriction site. DNA from the resulting plasmid pIC554(pCR rpoB01-ΔSma) (FIG. 2) was isolated for plastid transformation.

The Sma I restriction site of plasmid pIC571 enables easy one stepintegration of any foreign gene to be expressed in plastids.

Plastid Transformation of ΔrpoB Mutant Lines and Selection ofHomoplastomic Lines

The goal of the second transformation is to reconstitute the rpoB gene'sregulatory region (including the translation start), remove theaadA-cassette and introduce a marker restriction site at the same time.Young leaves from sterile homoplastomic ΔrpoB mutants grown onVBW-medium were bombarded with plasmid pIC554 coated gold particlesusing the Bio-Rad (Hercules, Calif., USA) PDS-1000/He Biolistic particledelivery system (for detailed procedure see example 3). Two days afterbombardment, leaves were cut into small pieces (ca. 3×3 mm) andtransferred to solid sucrose-reduced-RMOP medium (containing 3 g/litersucrose). Every three weeks the leaf pieces were cut again andtransferred to fresh medium until no further regenerates appeared. Thetransformants which display green phenotype and are able to growphotoautotrophically were selected and subjected to several additionalrounds of regeneration on sucrose-reduced-RMOP medium to obtainhomoplastomic tissue. Homoplastomic transplastomic lines were rooted andpropagated on solid B5-medium.

Molecular Analysis of the Secondary Transplastomic Plants

Total DNA isolated from sterile grown plants recovered from thesecondary transformation was used for DNA gel blot analysis.

The detailed procedure was as follows: 3 μg of total plant DNA peranalyzed plant were digested with restriction enzymes Bam HI and Sma Iat the same time and separated on a TAE agarose gel (0.8%). The DNA wasdenatured and transferred onto a positively charged nylon membrane(Hybond-N+, Amersham) as described in Ausubel et al. (1999). The filterwas hybridized with digoxigenin-labeled probes in DIG Easy Hyb Buffer(Roche Diagnostics GmbH, Mannheim, Germany), and hybridization signalswere detected using the DIG Luminescent Detection Kit (Roche). Themembrane was exposed to an X-OMAT LS film at room temperature for 75minutes.

For preparation of a hybridization probe, a 398 bp Sma I/Hind IIIfragment was excised from plasmid pCR rpoB01, purified from an agarosegel and labeled using the Dig probe labeling kit (Roche). This probeshould result in a signal of 3629 bp from the secondary transformedplastomes. This is a clear evidence, that the recombinant fragment fromthe transformation vector has been integrated, as a potentially wildtype derived band would have a size of only 1628 bp. The presence of the3629 bp fragment also indicates the removal of the aadA marker cassette.

To confirm the removal of the aadA marker a second hybridization of theblot (of which the former probe had been removed by a strippingprocedure) was done using a 480 bp fragment of the aadA-gene as probe.For probe generation primers oFCH59 and oFCH60 (see above) were used ina PCR Dig labeling reaction according to the protocol of the supplier(Roche).

Example 2 Construction of a Selection System Based on the Inactivationof the rpoA Gene

Construction of Transformation Vector pGEM-rpoA-del for the Inactivationof the rpoA Gene

The region of the tobacco chloroplast genome (corresponding to plastomenucleotides 79401–82470) containing the rpoA reading frame was amplifiedfrom genomic DNA isolated from leaf tissue of tobacco by PCR usingTaq-polymerase (Qiagen, Hilden, Germany). The following pair ofoligonucleotide primers was used: p78 5′-Sph I-TTA GTA ACA AGC AAA CCTTG-3′ (SEQ ID NO:6) (annealing with plastome nucleotides 79401–79420),and p77 5′-Sma I-TAA TTA CTG AAT CGC TTC CCA-3′ (SEQ ID NO:7) (annealingwith plastome nucleotides 82470–82450).

The PCR program used was as follows: 2 min at 94° C., 1 cycle; 45 sec at94° C., 45 sec at 55° C., 2 min at 72° C., 30 cycles; final extention at72° C. for 10 min. The fragment was ligated into the pGEM-T vector(Promega). The complete rpoA coding region (corresponding to plastomenucleotides 80455–81468) was subsequently deleted by digestion with DraI and Sca I. A chimeric aadA gene was excised from pUC16SaadA (for adetailed description of pUC16SaadA see Koop et al., 1996) as a Sma Ifragment and inserted to replace rpoA and to facilitate selection ofchlorolast transformations. A plasmid clone carrying the aadA gene inthe opposite orientation as rpoA yielded transformation vectorpGEM-rpoA-del (FIG. 3). The identity of the plasmid insert was verifiedby sequencing (MWG, Munich).

Primary Transformation and Selection of Homoplastomic ΔrpoA Mutants

Young leaves from sterile tobacco plants (cultivation see example 1)were bombarded with plasmid pGEM-rpoA-del coated gold particles usingthe Bio-Rad (Hercules, Calif., USA) PDS-1000/He Biolistic particledelivery system (detailed procedure see example 3). Two days afterbombardment, leaves were cut into small pieces (ca. 3×3 mm) andtransferred to solid RMOP-medium containing 500 μg/ml spectinomycin.Leaf pieces were cut again and transferred to fresh medium after 2weeks, then every 3 weeks until no further regenerants appeared. PrimaryΔrpoA transformants displayed spectinomycin-resistance and a greenphenotype in the light while still being heteroplastomic. In order toamplify transformed plastid DNA molecules and to eliminate wild-typegenomes, the primary transformants were subjected to 3 additional roundsof regeneration on selective medium. Since segregation leads to theoccurrence of white and green sectors, material from white sectors wassubjected to several additional rounds of regeneration on non-selectivemedium in order to obtain homoplastomic mutant transformants.Homoplastomic transformed lines were rooted and propagated on solidVBW-medium (Aviv and Galun, 1985; see example 1).

Molecular Analysis of Potential Plastid Transformants by SouthernAnalysis

3 μg of total plant DNA per analysed plant were digested with theappropriate restriction enzyme and separated on a TBE-agarose gel(0.8%). The DNA was denatured and transferred to a positively chargednylon membrane (Hybond-N+, Amersham) as described in Ausubel et al.,1999. The filter was hybridised with digoxigenin-labeled probes in DIGEasy Hyb Buffer (Roche Diagnostics GmbH, Mannheim, Germany), andhybridisation signals were detected using the DIG Luminescent DetectionKit (Roche). The membrane was exposed to a X-OMAT LS film at roomtemperature.

A fragment suitable for discrimination between wild type and transformedplastome was gel purified using the QIAquick Gel Extraction Kit (QIAgen,Hilden, Germany), labelled with digoxigenin using the Roche DIG DNALabelling Kit and used for hybridisation.

Construction of the Transformation Vector for Reconstitution of the rpoAGene

For a demonstration, that any gene of interest may be inserted using thedescribed selection system, a transformation vector was constructed,which reconstitutes the deletion of the rpoA coding region andintroduces a gus marker gene at the same time.

This vector contains the rpoA coding region and the gus gene, flanked by5′-and 3′-homologous sequences which were amplified from the tobaccochloroplast genome by PCR using the following two pairs of primers:oFCH112 5′-Nco I-TAC TAT TAT TTG ATT AGA TC-3′ (SEQ ID NO:8) (annealingwith plastome nucleotides 81471–81490), oFCH113 5′-Sma I-TAA TTA CTG AATCGC TTC CCA-3′ (SEQ ID NO:9) (annealing with plastome nucleotides82470–82450), and oFCH114 5′-Sph I-TTA GTA ACA AGC AAA CCT TG-3′ (SEQ IDNO:10) (annealing with plastome nucleotides 79401–79420), oFCH137 5′-PstI-ATC ACT AGT TGT AGG GAG GGA TCC ATG GTT CGA GAG AAA GTA AC-3′ (SEQ IDNO:11) (annealing with plastome nucleotides 81468–81449). The amplified5′-homologous fragment (corresponding to plastome nucleotides81471–82470) contains 1000 nucleotides upstream of the rpoA start codon.The amplified 3′-homologous fragment (corresponding to plastomenucleotides 79401–81468) contains a ribosome binding site (RBS), therpoA coding region, and 1054 nucleotides downstream of the rpoA stopcodon. The 5′ and 3′-homologous fragments are subcloned into plasmidpUC16SRBSuidA3′rbcL (Koop et al., 1996), regenerating transformationvector pIC598. The construction of this vector is shown in FIG. 4. Theidentity of the plasmid insert was verified by sequencing (MWG, Munich).

Plastid Transformation of ΔrpoA Mutant Lines and Selection ofHomoplastomic Lines

The goal of the second transformation is to reconstitute the rpoA codingregion, remove the aadA-cassette and introduce the gus marker gene atthe same time. Young leaves from sterile homoplastomic ΔrpoA mutantsgrown on VBW-medium were bombarded with plasmid pIC598-coated goldparticles using the Bio-Rad (Hercules, Calif., USA) PDS-1000/HeBiolistic particle delivery system (detailed procedure see example 3).Two days after bombardment, leaves were cut into small pieces (ca. 3×3mm) and transferred to solid sucrose-reduced-RMOP medium (containing 3g/liter sucrose). Every three weeks the leaf pieces were cut again andtransferred to fresh medium until no further regenerates appeared.Transformants which display green phenotype and are able to growphotoautotrophically were selected and subjected to several additionalrounds of regeneration on sucrose-reduced-RMOP medium to obtainhomoplastomic tissue. Homoplastomic transplastomic lines were rooted andpropagated on solid B5-medium.

Molecular Analysis of Potential Plastid Transformants by SouthernAnalysis

3 μg of total plant DNA per analysed plant were digested with theappropriate restriction enzyme and separated on a TBE-agarose gel(0.8%). The DNA was denatured and transferred to a positively chargednylon membrane (Hybond-N+, Amersham) as described in Ausubel et al.,1999. The filter was hybridised with digoxigenin-labelled probes in DIGEasy Hyb Buffer (Roche Diagnostics GmbH, Mannheim, Germany), andhybridisation signals were detected using the DIG Luminescent DetectionKit (Roche). The membrane was exposed to an X-OMAT LS film at roomtemperature.

A fragment suitable for discrimination between wild type and transformedplastome was gel purified using the QIAquick Gel Extraction Kit (QIAgen,Hilden, Germany), labelled with digoxigenin using the Roche DIG DNALabelling Kit and used for hybridisation.

Example 3 Construction of a White/Green Selection System Based onInactivation of the ycf3 Gene

Construction of Transformation Vector pIC553 for Targeted Inactivationof the ycf3

The region of the tobacco chloroplast genome containing the ycf3 readingframe was amplified from genomic DNA isolated from leaf tissue oftobacco by PCR using Taq-polymerase (Qiagen). The following pair ofoligonucleotide primers was used: oFCH63 (5′-GAA GTT TCT TTC TTT GCT ACAGC-3′ (SEQ ID NO:12), annealing with plastome nucleotides 45033–45053)and oFCH64 (5′-GM TTA CCA AAC CAT TTG ACC C-3′ (SEQ ID NO:13) annealingwith plastome nucleotides 47667–47647).

The PCR program used was as follows: 2 min at 94° C., 1 cycle; 45 sec at94° C., 45 sec at 55° C., 2 min at 72° C., 30 cycles; final extention at72° C. for 10 min. The fragment was ligated into the pGEM-T vector(Promega), regenerating plasmid pIC517. The first exon and 5′ regulatoryelement of ycf3 was subsequently deleted by digestion with Bbr PI andBst 11071. Bst 11071 cuts 373 nucleotides upstream of the ycf3 startcodon (nucleotide position 46266). The Bbr PI site is located withinintron 1 of ycf3 (close to the end of the first exon). A chimeric aadAgene was excised from pUC16SaadA (for a detailed description ofpUC16SaadA see Koop et al., 1996) as a Sma I fragment. It was insertedto replace ycf3 and to facilitate selection of plastid transformants. Aplasmid clone carrying the aadA gene in the opposite orientation as ycf3yielded transformation vector pIC553 (FIG. 5). The identity of theplasmid insert was verified by sequencing (MWG, Munich).

Electrotransformation of E. coli Cells

Preparation of electrocompetent cells: 1 liter of LB-medium (1% (w/v)casein hydrolysate, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl) isinoculated 1:100 with fresh overnight culture of E. coli JM109 cells(Promega, Madison, Wis., USA). The cells are grown at 37° C. withshaking at 220 rpm to an optical density of 0.5 at 600 nm. The cells arechilled on ice for 20 min and centrifuged for 15 min (4000 rpm, 4° C.).The supernatant is removed and the pellet is resuspended in 1 liter ofice-cold sterile 10% (v/v) glycerol. The cells are centrifuged two timesas described before, resuspending the cells in 500 ml and 20 ml ofice-cold sterile 10% (v/v) glycerol, respectively. The cells arecentrifuged an additional time and the pellet is resuspended in 2 ml ofice-cold sterile 10% (v/v) glycerol. This suspension is frozen inaliquots of 80 μl and stored at −80° C.

Electrotransformation using the Bio-Rad (Hercules, Calif., USA) MicroPulser electroporation apparatus: The electrocompetent cells are thawedon ice. 40 μl of the cell suspension are mixed with 2 μl of the ligationmixture and transferred into a prechilled, sterile 0.2 cm cuvette(Bio-Rad). The suspension is shaken to the bottom and the cuvette isplaced into the chamber slide. The chamber slide is pushed into thechamber and the cells are pulsed at 2.5 kV. The cuvette is removed fromthe chamber and the cells are suspended in 1 ml of SOC-medium (2% (w/v)casein hydrolysate, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂ and 20 mM glucose). The suspension is shaken for 1 h at 37° C.and 100 μl of the suspension is plated on LB plates containing 150 mg/lampicillin.

Primary Transformation and Selection of Homoplastomic Δycf3 Mutants

Tobacco seeds (Nicotiana tabacum cv. petit havanna) were surfacesterilized (1 min in 70% ethanol, 10 min in 5% Dimanin C, Bayer,Leverkusen, Germany), washed 3 times for 10 min in sterile H₂O and puton B5 medium (preparation see below). Plants were grown at 25° C. in a16 h light/8 h dark cycle (0.5–1 W/m², Osram L85W/25 Universal-Whitefluorescent lamps).

6 leaves from 4 weeks old, sterile grown Nicotiana tabacum L. var. petithavanna plants were cut and transferred on RMOP-medium (preparation seebelow). 35 μl of a gold suspension (0.6 micron, Biorad, München; 60mg/ml ethanol) was transferred into a sterile Eppendorf-cup (Treff,Fisher Scientific, Ingolstadt, Germany), collected by centrifugation andwashed with 1 ml sterile H₂O. The gold pellet was resuspended in 230 μlsterile H₂O, 250 μl 2.5 M CaCl₂ and 25 μg DNA (transformation vectorpIC553) were added. After thoroughly resuspending the mixture, 50 μl 0.1M spermidin were added, mixed and incubated for 10 min on ice. Then thegold was collected by centrifugation (1 min, 10000 rpm) and washed twicewith 600 μl ethanol (100%, p.A.). The gold was collected bycentrifugation (1 min, 10000 rpm) and finally resuspended in 72 μlethanol (100%, p.A.). A macrocarrier was inserted in the macrocarrierholder and 5.4 μl of the gold-suspension were applied. The bombardmentwas carried out with a Bio-Rad (Hercules, Calif., USA) PDS-1000/HeBiolistic particle delivery system using the following parameters:

rupture disc 900 psi

helium pressure 1100 psi

vacuum 26–27 inches Hg

macrocarrier at the top level

leaf piece at the third level

6 leaf pieces were bombarded each with 5.4 μl gold-suspension. Afterbombardment the leaf pieces were incubated for 2 days at 25° C. onRMOP-medium.

Two days after bombardment, leaves were cut into small pieces (ca. 3×3mm) and transferred to solid RMOP-medium containing 500 μg/mlspectinomycin. Leaf pieces were cut again and transferred to freshmedium after 2 weeks, then every 3 weeks until no further regeneratesappeared. Primary Δycf transformants displayed spectinomycin-resistanceand a green phenotype in the light while still being heteroplastomic. Inorder to amplify transformed plastid DNA molecules and to eliminatewild-type genomes, the primary transformants were subjected to 3additional rounds of regeneration on selective medium. Since segregationleads to the occurrence of white, mixed and green sectors, material fromwhite sectors was subjected to several additional rounds of regenerationon non-selective medium in order to obtain homoplastomic mutanttransformants. Homoplastomic transformed lines were rooted andpropagated on solid VBW-medium (Aviv and Galun, 1985) (preparation seebelow) under low light condition to obtain wild-type-similar Δycf3mutants (display light green phenotype).

RMOP (pH5.8 with KOH): NH₄NO₃(1650 μg/ml), KNO₃(1900 μg/ml), CaCl₂×2H₂O440 (μg/ml), MgSO₄×7H₂O (370 μg/ml), KH₂PO4 (170 μg/ml), EDTA-Fe(III)Na(40 μg/ml), KI (0.83 μg/ml), H₃BO₃ (6.2 μg/ml), MnSO₄×H₂O (22.3 μg/ml),ZnSO₄×7H₂O (8.6 μg/ml), Na₂MoO₄×2H₂O (0.25 μg/ml), CuSO₄×5H₂O (0.025μg/ml), CoCl₂×6H₂O (0.025 μg/ml), Inositol (100 μg/ml), Thiamine-HCl (1μg/ml), Benzylaminopurine (1 μg/ml), Naphthalene acetic acid (0.1μg/ml), Sucrose (30000 μg/ml), Agar, purified (8000 μg/ml).B5 (pH5.7 with KOH): KNO₃ (2500 μg/ml), CaCl₂×2H₂O (150 μg/ml),MgSO₄×7H₂O (250 μg/ml), NaH₂PO₄×H₂O (150 μg/ml), (NH₄)₂SO₄ (134 μg/ml),EDTA-Fe(III)Na (40 μg/ml), KI (0.75 μg/ml), H₃BO₃ (3 μg/ml), MnSO₄×H₂O(10 μg/ml), ZnSO₄×7H₂O (2 μg/ml), Na₂MoO₄×2H₂O (0.25 μg/ml), CuSO₄×5H₂O(0.025 μg/ml), CoCl₂×6H₂O (0.025 μg/ml), Inositol (100 μg/ml),Pyridoxine-HCl (1 μg/ml), Thiamine-HCl (10 μg/ml), Nicotinic acid (1μg/ml), Sucrose (20000 μg/ml), Agar, purified (7000 μg/ml).VBW (pH5.8 with KOH): NH₄NO₃(1650 μg/ml), KNO₃ 1900 (μg/ml), CaCl₂×2H₂O(440 μg/ml), MgSO₄×7H₂O (370 μg/ml), KH₂PO₄ (170 μg/ml), EDTA-Fe(III)Na(40 μg/ml), KI (0.83 μg/ml), H₃BO₃(6.2 μg/ml), MnSO₄×H₂O (22.3 μg/ml),ZnSO₄×7H₂O (8.6 μg/ml), Na₂MoO₄×2H₂O (0.25 μg/ml), CuSO₄×5H₂O (0.025μg/ml), CoCl₂×6H₂O (0.025 μg/ml), Inositol (100 μg/ml), Pyridoxin-HCL(0.5 μg/ml), Thiamine-HCl (1 μg/ml), Glycine (2 μg/ml), Nicotinic acid(0.5 μg/ml), Indolylacetic acid (2 μg/ml), Kinetin (0.2 μg/ml), Sucrose(30000 μg/ml), Caseinhydrolysat (500 μg/ml), Agar, purified (7000μg/ml).Analysis by PCR and Southern Blotting

Plastid transformants were identified by PCR amplification. Total DNAisolated from the first regenerates of 40 independent lines were used astemplates for separate PCR reactions. The method used was as follows:100 mg fresh leaf tissues of tobacco were disrupted (2×1 min at 25 Hz)in 200 μl AP1 buffer (DNeasy plant mini kit, QIAGEN)/1 μl reagent DX(foaming inhibition, QIAGEN) using mixer mill MM 300 (Retsch) in a 1.5ml microcentrifuge tube with one 3 mm tungsten carbide bead. DNA wasthen purified using the DNeasy plant mini kit. Five sets of primers(sequences are shown in table 1), namely oFCH59 and oFCH60; oFCH52 andoFCH53; oFCH52 and oFCH60; oFCH53 and oFCH59; oFCH60 and oFCH27 wereemployed to analyze transplastomic plants. oFCH52 and oFCH53 shouldresult in an amplification product of 900 bp from the wild-type plastomeand a product of 1700 bp from transformed plastomes, whereas oFCH59 andoFCH60 should result in an amplification product of 480 bp from thetransformed plants and no product from wild-type. Likewise, oFCH52,oFCH60 and oFCH53, oFCH59 should only amplify a product of 867 bp and1368 bp from the transformed plants, respectively. The combination ofoFCH60 and oFCH27 can determine whether the transformants carry correctinsertions or not by amplifying a product of 2541 bp from correctlytransformed plastomes.

TABLE 1 Primers sequences location oFCH59 5′-TGC TGG CCG TAC ATT TGTACG-3′ derived from the 5′ portion of the (SEQ ID NO:3) aadA codingregion oFOH60 5′-CAC TAC ATT TCG CTC ATC GCC-3′ derived from the 3′portion of the (SEQ ID NO:4) aadA coding region oFCH52 5′-CAC TAC ATTTCG CTC ATC GCC-3′ annealing with plastome nucleotides (SEQ ID NO:14)45903-45922, located within cloned plastid DNA fragment oFOH53 5′-GACTAT AGT TAA TGG ATA CTT-3′ annealing with plastome nucleotides (SEQ IDNO:15) 46812-46792, located within cloned plastid DNA fragment oFOH275′-TGC TCA AGA CTT TAG TGG ATC-3′ annealing with plastome nucleotides(SEQ ID NO:16) 44799-44819, located within chloroplast genome outside ofcloned plastid DNA fragment

PCR results showed that 24 lines of transformants carried the aadA genewith correct insertion in the plastid genome but they were stillheteroplastomic in the first cycle of regeneration. The data are alsoconsistent with phenotypic appearance of the respective lines, whichindicated that the pigment deficiency was correlated with deletion ofycf3.

Homoplasmy was verified by DNA gel blot analysis. Genomic DNAs isolatedfrom young leaves from plants derived from the fourth cycle ofregeneration grown under low light conditions were used for DNA gel blotanalysis. The detailed procedure was as follows: 4 μg of total plant DNAper analyzed plant were digested with restriction enzyme Xma JI andseparated on a TBE-agarose gel (0.8%). The DNA was denatured andtransferred to a positively charged nylon membrane (Hybond-N+, Amersham)as described in Ausubel et al. (1999). The filter was hybridized withdigoxigenin-labeled probes in DIG Easy Hyb Buffer (Roche DiagnosticsGmbH, Mannheim, Germany), and hybridization signals were detected usingthe DIG Luminescent Detection Kit (Roche). The membrane was exposed toan X-OMAT LS film at room temperature for 80 minutes.

For preparation of a DIG labeled probe, plasmid pIC522 (see below) wasused as template to amplify a 520 bp fragment using the following pairof primers: oFCH69 (5′-CAT TGG AAC TGC TAT GTA GGC-3′ (SEQ ID NO:17),corresponding to tobacco plastome sequence 47149–47169) and oFCH64(5′-GAA TTA CCA AAC CAT TTG ACC C-3′ (SEQ ID NO:13), corresponding totobacco plastome sequence 47667–47647). The PCR DIG Probe Synthesis Kitfrom Roche was used. The PCR program was as follows: 2 min at 94° C., 1cycle; 30 sec at 94° C., 30 sec at 55° C., 1 min at 72° C., 35 cycles;final extension at 72° C. for 10 min. The amplified fragment was gelpurified using the QIAquick Gel Extraction Kit Qiggen, Hilden, Germany)and then used for hybridization. This probe should result in a signal of2998 bp from the transformed plastomes and a signal of 2198 bp fromwild-type plastomes. The result showed that no wild-type plastid DNAcould be detected in all 10 examined mutant lines.

Construction of Transformation Vector pIC526 for Reconstitution of theycf3 Gene

Transformation vector pIC526 was designed to transform the mutant Dycf3line with the goal to reconstitute the ycf3 gene, delete the aadAcassette and insert a GFP gene at the same time.

The region of the tobacco chloroplast genome containing the first exonand 5′ regulatory element of ycf3 (571 bp) was amplified from genomicDNA isolated from leaf tissue of tobacco by PCR. The following pair ofoligonucleotide primers was used: oFCH48 5′-Sma I-Dra I-Kpn I-GTG TTTTTC TCC TCG TAA GAC-3′ (SEQ ID NO:18) (annealing with plastomenucleotides 46070–46090) and oFCH49 5′-Sma I-Bam HI-Bbr PI-Nhe I-CCG TTATGT ACA CAA AAT TG-3′ (SEQ ID NO:19) (annealing with plastomenucleotides 46637–46618). The PCR program was as follows: 2 min at 94°C., 1 cycle; 45 sec at 94° C., 45 sec at 55° C., 2 min at 72° C., 30cycles; final extension at 72° C. for 10 min. The fragment was digestedwith Sma I and ligated into plasmid pIC517 (construction see above)digested with Bbr Pi and Bst 11071. A plasmid clone carrying the firstexon and 5′ regulatory element of ycf3 in the correct orientationregenerated plasmid pIC522, which contains a cloned plastid DNA withadditional 5 restriction sites.

The coding region of GFP was amplified from plasmid pKCZ-GFP (FIG. 6) byPCR using the following pair of primers: oFCH25 (5′-CTA GCT AGC TTA TTTGTA TAG TTC ATC CAT-3′ (SEQ ID NO:20) and oFCH26 (5′-TCC CCC GGG GCC GTCGTT CAA TGA GAA TGG-3′ (SEQ ID NO:21). The PCR program was as follows: 2min at 94° C., 1 cycle; 45 sec at 94° C., 45 sec at 55° C., 2 min at 72°C., 30 cycles; final extension at 72° C. for 10 min. The amplified GFPfragment was cut with Sma I and Nhe I, and then ligated into pIC522 cutwith Bbr PI and Nhe I, generating pIC526 (FIG. 3). The identity of theplasmid insert was verified by sequencing (MWG, Munich).

Plastid Transformation of Dycf3 Mutant Lines and Selection ofHomoplastomic Lines

The goal of the second transformation was to reconstitute the ycf3 gene,remove the aadA marker and to introduce the gfp gene at the same time.Young leaves from sterile homoplastomic Dycf3 mutants grown under lowlight conditions on solid VBW medium were bombarded with plasmid pIC526coated gold particles using the Bio-Rad (Hercules, Calif., USA)PDS-1000/He Biolistic particle delivery system (detailed procedure seeabove). Two days after bombardment, leaves were cut into small pieces(ca. 3×3 mm), transferred to solid sucrose-reduced-RMOP medium(containing 3 g/liter sucrose) and cultivated under low light conditionsfor two weeks. Every three weeks leaf pieces were cut again, transferredto fresh medium and cultivated under strong light conditions until nofurther regenerates appeared. Transformants, which display a greenphenotype and are able to grow photoautotrophically were selected andsubjected to several additional rounds of regeneration onsucrose-reduced-RMOP medium to obtain homoplastomic tissue.Homoplastomic transplastomic lines were rooted and propagated on solidB5-medium under strong light condition.

Molecular Analysis of the Secondary Transplastomic Plants

Plastid transformants were identified by PCR amplification. Total DNAisolated from primary transformants which displayed green phenotype andwere able to grow photoautotrophically was used as a template for PCRanalysis using the following primer pair: oFCH76 (5′-GTA GCA ATC CAT TCTAGA AT-3′ (SEQ ID NO:22), annealing with plastome nucleotides46269–46288) and oFCH53 (5′-GAC TAT AGT TAA TGG ATA CTC-3′ (SEQ IDNO:15), annealing with plastome nucleotides 46812–46792). This pair ofoligonucleotide primers should result in an amplification product of 540bp from the wild-type plastome, a product of 1400 bp from plastomescorrectly transformed in the second round, and no product from unchangedfirst round transformants (since the site for p76 annealing wasdeleted).

Homoplasmy was verified by DNA gel blot analysis. Genomic DNA wasisolated from young leaves of plants derived from the fourth cycle ofregeneration grown under strong light conditions and digested with AvaI. The probe used was the same as that for Dycf3 mutants (detailedprocedures for DNA blotting and hybridization see above). The probegenerates a signal of 1212 bp for wild-type plastome, a signal of 2015bp for plastomes correctly transformed in the second round, and a signalof 6852 bp for unchanged first round transformants.

To confirm the removal of the aadA marker a second hybridization of theblot (of which the former probe had been removed by a strippingprocedure) was done using a 480 bp fragment of the aadA-gene as probe.For probe generation primers oFCH59 and oFCH60 (see above) were used ina PCR Dig labeling reaction according to the protocol of the supplier(Roche).

Example 4 Construction of a Selection System Based on the Inactivationof a Photosynthesis Related Gene

Construction of Transformation Vector pIC558 for Inactivation of thePlastid Encoded petA Gene

All cloning procedures were carried out using standard protocols asdescribed in example 1 and in Ausubel et al., 1999.

Vector pIC558 comprises two flanking sequences derived from the tobaccoplastome and an aadA-cassette (pUC16S aadA Sma vollst, Koop et al.,1996) in between. The homologous sequences are 5′ and 3′ regions of thepetA gene, 1 kb each. The aadA-cassette replaces the petA gene (962 bp)and 300 bp of the petA 3′ region.

Both flanking fragments were amplified by PCR using the following oligopairs as primers: oSK13 (5′-GGAATTCCATATGGTATAAAACTCATGTGTGTAAGAAA-3′)(SEQ ID NO:23) and oSK14 (5′-TCCCCCGGGGGTCCAATCATTGATCGCGAAA-3′) (SEQ IDNO:24), generating an Nde I and a Sma I site at the ends, and oSK15(5′-TTCCCCGGGTTCTAAATAGAAAGA AAGTCAAATTTG-3′) (SEQ ID NO:25) and oSK16(5′-CATGCATGCGAATGAATAAGATTCTCTTAGCTC-3′) (SEQ ID NO:26), generating aSma I and a Sph I site at the fragment ends. The PCR program used was asfollows: 3 min at 94° C., 1 cycle; 45 sec at 94° C., 45 sec at 55° C.,1.5 min at 72° C., 30 cycles; final extension at 72° C. for 10 min. Thedigested fragments (left/right flank) and the aadA-cassette as Sma Ifragment were cloned in one step into the pUC19 vector which wasdigested with Nde I and Sph I. Construct pIC558 was analyzed byrestriction experiments. The PCR amplified fragments were sequenced toprove the correct sequence of the flanking regions.

Transformation Vector pIC558 is shown in FIGS. 8 and 9.

Construction of Transformation Vector pIC597, pIC599 and pIC600 forReconstitution of the petA Gene

The aim of the second transformation is to cure the petA inactivationand insert a new gene of interest (uidA or aphA-6, potentialy npt II)into the plastome simultaneously. Therefore, the petA gene and a genecassette (containing 5′/3′ regulatory elements) were cloned in betweenthe left/right flanking sequences. Vector pIC597 (uidA-cassette)comprises the same flanking sequences as vector pIC558, the petA geneand the uidA gene-cassette.

A fragment of about 2.2 kb containing 1 kb left flank, the petA genesequence (962 bp) and 300 bp of the 3′ region of the petA gene wasamplified by PCR using the following oligo pair as primers: oSK13(5′-GGAATTCCATATGGTATAAAACTCATGTGTGTAAGAAA-3′) (SEQ ID NO:23) and oSK71(5′-TCCCCCGGGTAGAAAACTATTGATACGTCTTATGG-3′), (SEQ ID NO:27), generatingan Nde I and a Sma I site at the fragment ends. The PCR program used wasas follows: 3 min at 94° C., 1 cycle; 45 sec at 94° C., 45 sec at 55°C., 3 min at 72° C., 30 cycles; final extension at 72° C. for 10 min.This fragment and the right flank were cloned together into pUC19. Thisvector pIC651 (‘petA+1 kb5’+1,3 kb3″) comprises a 1 kb left flank, thepetA coding sequence, 300 bp of the 3′ region and a 1 kb right flankcorresponding to Nicotiana tabacum plastome sequence 63.335–66.597.

The new gene of interest (either uidA, Koop et al., 1996; or aphA-6,vector pSK.KmR, Bateman and Purton, 2000; or npt II, Töpfer et al.,1987) was introduced as gene cassette (containing 5′/3′ regulatoryelements) between both flanking fragments. The uidA-cassette (as Sma Ifragment) was taken from vector pIC562 (‘pUC16SRBSuidA3′rbcL’, Koop etal., 1996). The genes aphA-6 and npt II were cloned into vector pIC562replacing the uidA-gene, each. After this cloning step theaphA-6-cassette and an npt II-cassette could be isolated by Sma Idigestion, respectively. These cassettes were cloned into the petA 3′region (insertions site 300 bp downstream to petA). These vectors arenamed ‘petA-cure-plasmids’ (pIC597 with uidA; pIC599 with aph6; pIC600with nptII).

The constructs were analyzed by restriction experiments and PCRamplified fragments were sequenced to prove the correct sequence of theflanking regions.

A schematic representation of the three vectors is given in FIG. 10.Transformation vector pIC597 is shown in FIG. 11.

Primary Transformation and Selection of Homoplastomic DpetA Mutants.

Plastid transformation by particle gun with vector pIC558 and selectionwas carried out as described in example 3. PEG mediated plastidtransformation with vector pIC558 and selection was carried out asdescribed in example 1.

Secondary Transformation and Selection of Reconstituted HomoplastomicDpetA Mutants.

Plastid transformation by particle gun with vector pIC558 was carriedout as described in example 3. PEG mediated plastid transformation withvector pIC588 was carried out as described in example 1. Selection oftransformants was done

-   a) on RMOP medium with reduced sucrose content (0.3%). Transformants    with a reconstitution of the petA knockout should be able to use    photosynthetic energy for growing.-   b) on RMOP medium containing kanamycin as selection agent (gene    products of aph-6 and nptII detoxify kanamycin).    Transformants showed a decrease of hcf (high chlorophyll    fluorescence) during repeated cycles of regeneration.    Analysis of Transformants by PCR and Southern Blotting After Primary    Transformation

For plant DNA isolation, PCR analysis and Southern blotting standardprotocols were used as described in Example 1. For determination of theaadA gene, primers oFCH59aadA480-li and oFCH60-aadA480-re (5′-CAC TACATT TCG CTC ATC GCC-3′) (SEQ ID NO:4) were used. To determine whetherthe transformants carry correct insertions, primers oFCH60-aadA480-reand oSK116-petA-re (5′-AAAATAGATTCATTAGTCCGATACC-3′) (SEQ ID NO:28) wereused. Primer oSK116-petA-re is located upstream (outside) of the 5′flanking fragment. The PCR program used was as follows: 3 min at 94° C.,1 cycle; 45 sec at 94° C., 45 sec at 55° C., 2 min at 72° C., 30 cycles;final extension at 72° C. for 10 min.

First PCR results showed that 12 lines of transformants are carrying theaadA gene with correct insertion in the plastid genome. Further testingand southern analysis to show whether the lines are homoplastomic orheteroplastomic are carried out as described in example 1.

Analysis of Transformants by PCR and Southern Blotting After SecondaryTransformation

For plant DNA isolation and PCR analysis standard protocols were used asdescribed in example 1. For determination of the uidA gene primers,oSM61-GUS-N (5′-TCACACCGATACCATCAGCG-3′) (SEQ ID NO:29) and oSM62-GUS-C(5′-ATTGTTTGCCTCCCTGCTGC-3′) (SEQ ID NO:30) were used. To determinewhether the transformants carry correct insertions, primers oSM61-GUS-N(5′-TCACACCGATACCATCAGCG-3′) (SEQ ID NO:29) and oSK138-petA-3′-re(5′-AATCGTAACCAGTC TCTACTGG-3′) (SEQ ID NO:31) were used. The PCRprogram used was as follows: 3 min at 94° C., 1 cycle; 45 sec at 94° C.,45 sec at 55° C., 2 min at 72° C., 30 cycles; final extension at 72° C.for 10 min.

For detection of the aph-6 gene and the nptII gene specific primers wereused. To determine whether the transformants carry correct insertionsone gene specific primer and primer oSK138-petA-3′-re are used.

Southern blotting analysis are carried out as described in example 1 andin standard protocols.

Example 5 Selection for Paraquat Tolerance

Plant Transformation and Selection for Paraquat Resistance

4 leaf pieces were transformed each with 1 μg pIC558 (FIG. 8) asdescribed in example 4. After bombardment the leaf pieces were incubatedfor 2 days at 25° C. on RMOP-medium.

Two days after bombardment leaves were cut into small pieces (ca. 3×3mm), transferred to fresh RMOP-medium and incubated for 10 days in thedark at 25° C. Then leaf pieces were cut again, transferred to freshmedium containing 5 mg/l paraquat and incubated for 10 days in the lightat 25° C. The leaf pieces were cut again, transferred to fresh mediumcontaining 8 mg/l paraquat and incubated for 12 days in the light at 25°C. Green regenerates from the bottom side were retrieved and transferredto individual plates containing RMOP with 8 mg/l paraquat. The lineswere subjected to repeated cycles of shoot generation by cutting smallleaf pieces, which form new regenerates on RMOP-medium with 8 mg/lparaquat.

Molecular Analysis of Potential Plastid Transformants by SouthernAnalysis

3 mg of total plant DNA per analysed plant are digested with theappropriate restriction enzyme and separated on a TBE-agarose gel (1%).The DNA is denatured and transferred to a positively charged nylonmembrane (Hybond-N+, Amersham) as described in Ausubel et al., 1999:Short protocols in molecular biology, Wiley, 4^(th) edition, Unit 2.9A.The filter is hybridised with digoxigenin-labelled probes in DIG EasyHyb Buffer (Roche Diagnostics GmbH, Mannheim, Germany), andhybridisation signals are detected using the DIG Luminescent DetectionKit (Roche). The membrane is exposed to a X-OMAT LS film at roomtemperature.

A fragment suitable for discrimination between wild type and transformedplastome is gel purified using the QIAquick Gel Extraction Kit (QIAgen,Hilden, Germany), labelled with digoxigenin using the Roche DIG DNALabelling Kit and used for hybridisation.

Example 6 Reconstitution of ycf3 Using Kanamycin Selection

Construction of Transformation Vector pIC577 for Targeted Inactivationof the ycf3 Gene

A transformation vector was constructed designed to inactivate the ycf3gene by replacing the first exon and the splicing site of ycf3(corresponding to plastome nucleotides 46042–46206) with the aadA codingregion. This vector does not contain any 3′ regulatory elements (neitherfor the aadA marker gene, nor for the endogenous ycf3 or tRNA gene). Inaddition, no promoter elements were introduced, and the aadA gene isexpected to be transcribed and translated by the endogenous ycf3upstream regulatory element.

This vector contains the aadA coding region, flanked by 5′- and3′-homologous sequences which were amplified from the tobaccochloroplast genome by PCR using the following two pairs of primers:oFCH76 (5′-Nco I-GTA GCA ATC CAT TCT AGA AT-3′, (SEQ ID NO:22),annealing with plastome nucleotides 46269–46288) and oFCH77 (5′-SmaI-CGG AAA GAG AGG GAT TCT AAC-3′, (SEQ ID NO:32), annealing withplastome nucleotides 47205–46185); oFCH78 (5′-Sph I-GAA GTT TCT TTC TTTGCT ACA-3′ (SEQ ID NO:33), annealing with plastome nucleotides45033–45053) and oFCH79 (5′-Pst I-TAC GCT TTT T GA AGG TGA AGT-3′ (SEQID NO:34), annealing with plastome nucleotides 46041–46021).

The PCR amplification using Pfu polymerase (Promega) was performed asfollows: 2 min at 94° C., 1 cycle; 45 sec at 94° C., 45 sec at 55° C., 2min at 72° C., 30 cycles; final extention at 72° C. for 10 min. Theamplified 5′-homologous fragment (corresponding to plastome nucleotides46269–47205), containing 936 nucleotides upstream of the ycf3 startcodon, was digested with Sma I and Nco I and then ligated intopUC16SaadA plasmid (Koop et al., 1996) which was digested with Eco RI,followed by a fill-in reaction using Klenow polymerase (Promega) andthen digested with Nco I, generating pIC565. The amplified 3′-homologousfragment (corresponding to plastome nucleotides 45033–46041), containing1000 nucleotides of the ycf3 gene, was digested with Pst I and Sph I,and then ligated into pIC565 cut with Pst I and Sph I, yielding thefinal transformation vector pIC577 (FIGS. 12 and 13). The identity ofthe plasmid insert was verified by sequencing (MWG, Munich).

Primary Transformation and Selection of Homoplastomic Δycf3 Mutants

Young leaves from sterile tobacco plants (cultivation see example 3)were bombarded with plasmid pIC577-coated gold particles using theBio-Rad (Hercules, Calif., USA) PDS1000/He Biolistic particle deliverysystem (detailed procedure see example 3). Two days after bombardment,leaves were cut into small pieces (ca. 3×3 mm) and transferred to solidRMOP-medium containing 500 μg/ml spectinomycin. Leaf pieces were cutagain and transferred to fresh medium after 2 weeks, then every 3 weeksuntil no further regenerants appeared. Primary Δycf3 transformantsdisplayed spectinomycin-resistance and a green phenotype in the lightwhile still being heteroplastomic. In order to amplify transformedplastid DNA molecules and to eliminate wild-type genomes, the primarytransformants were subjected to 3 additional rounds of regeneration onselective medium. Since segregation leads to the occurrence of white andgreen sectors, material from white sectors was subjected to severaladditional rounds of regeneration on non-selective medium in order toobtain homoplastomic mutant transformants. Homoplastomic transformedlines were rooted and propagated on solid VBW-medium (Aviv and Galun,1985; see example 3).

Analysis by PCR and Southern Blotting

Plastid transformants were identified by PCR amplification. The totalDNA isolated from the first regenerates of 24 independent lines wereused as a template for PCR. Two sets of primers (the sequences seeexample 3): oFCH59 and oFCH60; oFCH52 and oFCH53 were employed toanalyze transplastomic plants. oFCH52 and oFCH53 should result in anamplification product of 900 bp from the wild-type plastome and aproduct of 1476 bp from transformed plastomes, whereas oFCH59 and oFCH60should result in an amplification product of 480 bp from the transformedplants and no product from wild-type. The results show that 14 lines oftransformants carry correct aadA insertions in the plastid genome. Thedata are also consistent with phenotypic appearance of the respectivelines, which indicated that the pigment deficiency was correlated withdeletion of ycf3.

Homoplasmy was verified by DNA gel blot analysis. Genomic DNAs isolatedfrom young leaves of Δycf3 mutants (4^(th) regenerates) grown under lowlight conditions were used for DNA gel blot analysis. Detailed procedurewas as follows: 4 μg of total plant DNA per analyzed plant was digestedwith restriction enzyme Xma JI and separated on a TAE-agarosegel (0.8%).The DNA was denatured and transferred to a positively charged nylonmembrane (Hybond-N⁺, Amersham) as described in Ausubel et al. (1999).The filter was hybridized with digoxigenin-labeled probes in DIG EasyHyb Buffer (Roche Diagnostics GmbH, Mannheim, Germany), andhybridization signals were detected using the DIG Luminescent DetectionKit (Roche). The membrane was exposed to a X-OMAT LS film at roomtemperature for 2 hours.

For preparation of a DIG-labeled probe, tobacco genomic DNA was used astemplate to amplify a 520 bp fragment using the following pair ofprimers: oFCH69 (5′-CAT TGG AAC TGC TAT GTA GGC-3′ (SEQ ID NO:17),corresponding to tobacco plastome sequence 47149–47169) and oFCH64(5′-GAA TTA CCA AAC CAT TTG ACC C-3′ (SEQ ID NO:13),corresponding totobacco plastome sequence 47667–47647). The PCR DIG Probe Synthesis Kitfrom Roche was used. The PCR program was as follows: 2 min at 94° C., 1cycle; 30 sec at 94° C., 30 sec at 55° C., 1 min at 72° C., 30 cycles;final extension at 72° C. for 10 min. The amplified fragment was gelpurified using the QIAquick Gel Extraction Kit Qiagen, Hilden, Germany)and then used for hybridization. This probe should result in a signal of2780 bp from the transformed plastomes and a signal of 2198 bp fromwild-type plastomes. The result showed that no wild-type plastid DNAcould be detected in all 6 examined mutant lines.

Construction of the Transformation Vector pIC637 for Reconstitution ofthe ycf3 Gene

Transformation vector pIC637 was designed to transform the mutant Δycf3line with the goal to reconstitute the ycf3 gene, delete the aadA geneand insert the aphA-6 gene that confers resistance to kanamycin at thesame time.

The aphA-6 gene is introduced into the upstream position of ycf3 withoutdisruption of either ycf3 expression or the function of the endogenousycf3 upstream regulatory element. A short RBS (ribosomal bonding site)sequence serves as the signal to translate the reconstituted ycf3 geneas a newly formed artificial operon. The aphA-6 gene and ycf3 aretranscribed in the same direction under control of ycf3 5′-regulatoryelement.

The region of the tobacco chloroplast genome (corresponding to plastomenucleotides 45033–46266) containing the N-terminal of ycf3 (which isdeleted in the first round transformation) was amplified from genomicDNA isolated from leaf tissue of tobacco by PCR. The following pair ofoligonucleotide primers were used: oFCH139 (5′-Pst I-ATC ACT AGT TGT AGGGAG GGA TCC (ribosome binding site)-ATG CCT AGA TCA CGG ATA AA-3′ (SEQID NO:35), annealing with plastome nucleotides 46266–46247) and oFCH78(5′-Sph I-GAA GTT TCT TTC TTT GCT ACA-3′ (SEQ ID NO:33), annealing withplastome nucleotides 45033–45053). The PCR amplification using Taqpolymerase (Promega) was performed as follows: 2 min at 94° C. 1 cycle;45 sec at 94° C., 45 sec at 55° C., 2 min at 72° C., 30 cycles; finalextension at 72° C. for 10 min. The fragment was digested with Pst I andSph I, and then ligated into pIC577 cut with Pst I and Sph I, generatingpIC636.

The coding region of the aphA-6 gene was cut from the plasmid pSK.KmR(obtained from Dr. Saul Purton, Department of Biology University collegeLondon, UK) using Nco I and Pst I and then ligated into pIC636 cut withNco I and Pst I, yielding the final transformation vector pIC637 (FIGS.14 and 15). The identity of the plasmid insert was verified bysequencing (MWG, Munich).

Plastid Transformation of Δycf3 Mutant Lines and Selection ofHomoplastomic Lines

The goal of the second transformation was to reconstitute the ycf3 gene,remove the aadA marker and introduce the aphA-6 gene that confersresistance to kanamycin at the same time. Embedded protoplasts isolatedfrom sterile homoplastomic Δycf3 mutants grown under low lightconditions on solid VBW-medium were bombarded with plasmid pIC637-coatedgold particles using the Bio-Rad (Hercules, Calif., USA) PDS-1000/HeBiolistic particle delivery system (detailed procedure see example 3).Two days after bombardment, grids were transferred to solid RMOP medium,containing 25 μg/ml kanamycin and cultivated under low light conditionsfor two weeks. Afterwards, every two weeks grids were transferred tofresh medium and cultivated under strong light conditions until nofurther regenerates appeared. The transformants which display kanamycinresistance and a green phenotype were selected and subjected to B5medium under strong light condition to amplify ycf3-reconstitutedplastomes (ycf3-deficient plastomes can not be amplified when growing onB5 medium and strong light conditions).

Molecular Analysis of the Secondary Transplastomic Plants

Plastid transformants were identified by PCR amplification. The totalDNA isolated from primary transformants which displayed green phenotypeand were able to grow photoautotrophically was used as a template forPCR analysis using the following two pairs of primers: oFCH168 (5′-TCAGTC GCC ATC GGA TGT TT-3′ (SEQ ID NO:36), derived from the 5′ portion ofthe aphA-6 coding region) and oFCH169 (5′-ACC AAT CTT TCT TCA ACA CG-3′(SEQ ID NO:37), derived from the 3′ portion of the aphA-6 codingregion); oFCH27 (5′-TGC TCA AGA CTT TAG TGG ATC-3′ (SEQ ID NO:16),annealing with plastome nucleotides 44799–44819) and oFCH168. oFCH168and oFCH169 should result in an amplification product of 500 bp from thereconstituted plants and no product from unchanged first roundtransformants. The combination of oFCH27 and oFCH168 can determinewhether the second round transformants carry correct aphA-6 insertionsor not by amplifying a product of about 2300 bp from correctlytransformed plastomes. In total, 5 unique ycf3-reconstituted tobaccoplastid transformants were obtained from 3 grid bombardments.

Homoplasmy was verified by DNA gel blot analysis. Genomic DNA wasisolated from young leaves of ycf3-reconstituted plants grown on B5medium under strong light conditions and digested with Hinc II. Theprobe used was the same as that for Δycf3 mutants (detailed proceduresfor DNA blotting and hybridization see above). The probe generates asignal of 3257 bp for wild-type plastome, a signal of 2046 bp forplastomes correctly transformed in the second round, and a signal of3857 bp for unchanged first round transformants.

To confirm the removal of the aadA marker a second hybridization of theblot (of which the former probe had been removed by a strippingprocedure) was done using a 480 bp fragment of the aadA-gene as probe.For probe generation primers oFCH59 and oFCH60 (see above) were used ina PCR DIG labeling reaction according to the protocol of the supplier(Roche).

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1. A process for producing a multicellular dicotyledonous plant, plantorgan or plant tissue having a transformed plastome, wherein the plant,plant organ or plant tissue is selected from the group consisting ofpotato, tomato and tobacco, the process comprising the following steps:(a) altering or disrupting the function of a gene in a plastid genomefor producing a selectable or recognizable phenotype, wherein saidphenotype is a pigment deficiency and/or photosynthesis deficiency; (b)separating or selecting a plant, plant organ or plant tissue havingplastids expressing said phenotype from plants, plant organs or planttissues having plastids that do not express said phenotype, therebyproducing a separated or selected plant, plant organ or plant tissuehaving plastids expressing said phenotype; (c) transforming said plastidgenome of said separated or selected plant, plant organ or plant tissueexpressing said phenotype with at least one transformation vector havinga restoring sequence capable of restoring said function; and (d)separating or selecting said transformed plant, plant organ or planttissue having plastids expressing said restored function fromnon-transformed plant, plant organ or plant tissue which does notexpress said restored function.
 2. The process according to claim 1,wherein the transformation of step (c) restores said function inconjunction with introducing at least one additional function.
 3. Theprocess according to claim 1, wherein the transformation of step (c)restores said function in conjunction with causing a desired additionalgenetic modification of the plastid genome.
 4. The process according toclaim 1, wherein the transformation of step (c) additionally eliminatesa preexisting function in another gene in said plastid genome of saidseparated or selected plant, plant organ or plant tissue.
 5. The processaccording to claim 1, wherein said alteration or disruption of step (a)is obtained by induced mutation.
 6. The process according to claim 1,wherein said alteration or disruption of step (a) is obtained by genetictransformation.
 7. The process according to claim 6, wherein saidgenetic transformation results simultaneously in the introduction of atleast one additional sequence for at least one additional function. 8.The process according to claim 7, wherein said additional function is aninhibitor resistance function and step (b) is carried out in thepresence of the corresponding inhibitor.
 9. The process according toclaim 8, wherein the inhibitor present in step (b) is not present duringregeneration.
 10. The process according to claim 1, wherein step (a)alters or disrupts a trophic type and step (c) restores a trophic type.11. The process according to claim 10, wherein the restored trophic typeis phototrophy.
 12. The process according to claim 1, wherein step (c)restores a deficiency produced in step (a).
 13. The process according toclaim 1, wherein the altering or disrupting of gene function in step (a)is done using a vector, further wherein the vector(s) used in step (a)and/or or (c) comprise(s) a sequence having homology to a host plastidsequence sufficient for homologous recombination.
 14. The processaccording to claim 1, wherein the plant, plant organ, or plant tissue instep (b) is grown or cultured in a medium supporting heterotrophicgrowth.
 15. The process according to claim 1, wherein said alteration ordisruption of step (a) produces a pigment deficient phenotype.
 16. Theprocess according to claim 15, wherein said pigment deficient phenotypeis chlorophyll deficiency.
 17. The process according to claim 1, whereinthe gene function altered or disrupted in step (a) is the function of aplastome encoded plastid gene essential for transcription ortranslation.
 18. The process according to claim 1, wherein the genefunction altered or disrupted in step (a) is the function of a plastidrpoA or rpoB gene.
 19. The process according to claim 1, wherein thephenotype produced in step (a) can alternate between two or severalappearances dependent on external growth conditions.
 20. The processaccording to claim 19, wherein the gene function altered or disrupted instep (a) is the function of a plastid ycf3 gene, producing ayellow-white phenotype under standard light conditions and a light greenphenotype under low light conditions.
 21. The process according to claim1, wherein the gene function altered or disrupted in step (a) is thefunction of a plastid gene which produces a high chlorophyllfluorescence phenotype upon alteration or disruption, preferably petA.22. The process according to claim 21, wherein the separation orselection in step (b) utilizes an inhibitor that requires activephotosynthesis for efficacy.
 23. The process according to claim 22,wherein said inhibitor is paraquat, morphamquat, diquat, difenzoquatand/or cyperquat.
 24. The process according to claim 1, whereinphotomixotrophic conditions are used in step (d).
 25. The processaccording to claim 1, wherein the altering or disrupting of genefunction in step (a) and the restoring of said gene function in step (c)comprises the introduction of a sequence in step (a) and theintroduction of a sequence in step (c), further wherein the sequenceintroduced in step (a) and the sequence introduced in step (c) togetherresult in an additional function.
 26. The process according to claim 1,wherein the separating or selecting of said plant, plant organ or planttissue in step (d) further comprises separating or selecting a plant,plant organ or plant tissue exhibiting resistance to an inhibitor from aplant, plant organ or plant tissue which does not exhibit resistance tothe inhibitor.
 27. The process according to claim 1, wherein the genefunction altered or disrupted in step (a) is the function of a plastidpsbA gene.